Antibodies are plasma proteins produced by the immune system as a response to foreign substances in the body, and are used for a wide variety of diagnostic, therapeutic, and research applications. For example, antibodies may be used in ELISA assays for detection and diagnosis of analytes and markers, as components of biosensors, as imaging agents, as pharmaceuticals, and in countless other uses. In the last fifteen years, monoclonal antibodies have been developed to be used as therapeutics to treat many human diseases, including various cancers and autoimmune diseases. Due to their high level of specificity and selectivity toward known targets, antibodies produce few nonspecific interactions, which reduce the side effects associated with many treatments.
Typically, antibodies are obtained through the purification of biological fluids, such as plasma, cell lysates, ascites fluid, and in the case of monoclonal antibodies produced in cell culture from hybridoma cells, from cell culture supernatant. Antibody purification may include any of a number of processes, such as ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography. Existing processes for purifying antibodies involve multiple steps that may have detrimental effects on the specific activity of isolated antibodies. For example, during purification, several processes may result in low yields and defective antibodies, including protein unfolding, misfolding, and aggregation; covalent modification (e.g., oxidation); enzymatic proteolysis; and the “scrambling” of light chains. Procedures for purifying antibodies generally are designed to remove a number of contaminants, such as host cell proteins, DNA, endotoxins, and cell culture media additives. In addition, antibody-derived impurities, such as high-molecular-weight aggregates and proteolytic fragments of immunoglobulin, may also contaminate the desired product.
Affinity chromatography is useful because it allows the possibility of obtaining several fold purification with high recovery in a single step. Protein A and protein G are the most commonly used capture proteins in the purification of human antibodies. However, protein A and protein G have several drawbacks, including their high cost, low stability, and the possibility of contaminating the product through hydrolysis and release of peptide fragments.